The present invention relates to saccharification enzymes (i.e., enzymes that hydrolyze polysaccharides into constituent sugars) obtainable from hyperthermophilic bacteria, and processes for producing them. In particular, this invention relates to the following types of thermostable enzymes: amylases, pullulanases, xcex1-glucosidases and xcex2-glucosidases.
Hyperthermophilic bacteria, i.e. bacteria which thrive on temperatures around the boiling point of water, are found in the depths of the ocean close to geothermal springs. Since these bacteria live in high temperature environments, their enzymes, which are essential to sustaining life processes, such as digestion and respiration, must be able to function at such extreme temperature conditions. Enzymes in common mesophilic bacteria (i.e., organisms that can grow at intermediate temperatures compared to the upper and lower extremes for all organisms) degenerate rapidly at these high temperatures.
Proteins which can function at high temperatures can be extremely advantageous for use in a number of industries. For example, soda syrup, laundry detergent, and many pharmaceuticals contain, or are manufactured by, enzymes extracted from microbes. If enzymes from hyperthermophilic bacteria were used in place of the enzymes commonly used today, the processes to make these products could be performed at higher temperatures. Higher temperatures speed up reactions and prevent contamination by fungi and common bacteria. Alternatively, lesser amounts of the enzymes from hyperthermophilic bacteria might be required to sustain enzymatic processes under current temperature conditions where the thermostability of such enzymes correlates with a longer useful life under those conditions.
A number of microorganisms that are capable of growth at or above 100xc2x0 C. (i.e., hyperthermophiles) have been isolated from several terrestrial and marine environments and are of considerable scientific interest. (See Kelly, R. M., and J. W. Deming, 1988, xe2x80x9cExtremely Thermophilic Archaebacteria: Biological and Engineering Considerationsxe2x80x9d, Chem. Engr. Prog. 4:47-62; and Wiegel J., and L. G. Ljungdahl, 1986, xe2x80x9cThe Importance of Thermophilic Bacteria In Biotechnology,xe2x80x9d CRC Crit. Rev. Microbiol. 3:39-107.) However, previously it has not been possible to take full advantage of the utility potential of these organisms, in large part because of a lack of understanding of their growth and metabolic characteristics.
Detailed study of specific enzymes from hyperthermophiles is just beginning, and the few known reports on such enzymes that have been published so far all appeared in 1989. For example, it has been shown (Pihl, T. D. et al., 1989, Proc. Nat. Acad. Sci. 86:138-141) that an extremely thermostable hydrogenase isolated from Pyrodictium brockii is immunologically related to the comparable enzyme in the Bradyrhizobium japonicum, a mesophile. Adams and coworkers have described several distinctive characteristics of a hydrogenase (Bryant, F. O. and Adams, M. W. W., 1989, J. Biol. Chem. 264:5070-5079) and a ferredoxin (Aono, S., et al., 1989, J. Bacteriol. 171:3433-3439) from the hyperthermophilic bacterium, Pyrococcus furiosus. 
In the broad context of the present invention, saccharification is the process of degradation of a polysaccharide into its basic constituents which are simple sugars. Complex polysaccharides, such as cellulose (in plant cell walls), chitin (in the outer coverings of insects and crustaceans), glycogen (in animal cells) and starch (in plant cells) are all macromolecular polymers of simple sugars, mainly D-glucose, coupled by various chemical linkages into linear and cross-linked or branched networks. Thus, to degrade such polymeric substrates into their simplest sugar components requires a variety of saccharification enzymes having different specificities with respect to the type of sugar (e.g, five or six carbon ring), the positions of linkages between those sugars [e.g., carbon 1 to carbon 4 (i.e., 1,4 linkage) or 1,6 linkage] and the isomeric configurations of those linkages (e.g., xcex1-1,4 or xcex2-1,4).
Starch, for example, is a highly branched polymer essentially composed of xcex1-D-glucose coupled by both xcex1-1,4- and xcex1-1,6- glycosidic linkages. The current industrial processes of digestion of starch into simple sugars (e.g., in the making of corn syrup sweeteners) illustrate many problems with the enzymes that are presently available for large scale digestion of polysaccharides. Two enzymatic steps are applied in the hydrolysis of starch to xcex1-D-glucose. Although the entire process may be considered saccharification in the broad context of this application, the two steps are commonly known as liquification and saccharification. Liquification involves the use of an xcex1-amylase to hydrolyze starch granules which have been slurried in water and gelatinized by heat to allow the enzyme to attack. This process is designed to reduce the viscosity of the hydrolysate and produce a high yield of low molecular weight polysaccharides, primarily dextrins. In the saccharification step, these dextrins are then further hydrolyzed to glucose by another enzyme activity which may be a classified as a glucoamylase. The commercial objective in the overall starch hydrolysis process is to obtain a hydrolysate with maximum glucose content (DX), at least 96% of the dry weight of starch.
A recent comparison of xcex1-amylases (i.e., 1,4-xcex1-D-glucanohydrolases) that are available for commercial scale starch liquification (Shetty, J. K. and Allen, W. G., 1988, Cereal Foods World 33:929-934) pointed out deficiencies in the industry standard, the Bacillus licheniformis xcex1-amylase, even though it does meet present operational and performance demands of starch processors. Areas for improvement include: reducing demands for chemicals, ion exchange processing, and other product refining; reducing by-product and color formation; and improving glucose yield and product quality.
Control of pH is critical to the efficiency and operation of current commercial starch liquification processes. Values of lower than pH 6.2 to 6.4 decrease the reaction rate and the stability of the Bacillus licheniformis xcex1-amylase. At higher. pH values, color and by-product formation during liquefaction increase with increasing pH. The pH of the refined starch slurry that enters the enzyme liquefaction process from the wet milling operation is normally in the range of 4.0-5.0. For liquification by the Bacillus licheniformis xcex1-amylase, base must be added to increase the pH of the starch slurry to the range of 6.2-6.4. After liquefaction, acid must be added for efficient saccharification, typically by a glucoamylase at pH 4.2-4.5. Therefore, the ability to perform liquefactions at a lower pH would decrease the demand for chemically adjusting pH prior to and after liquefaction. It would also reduce color and by-product formation, and lower refining requirements and costs.
An additional problem with Bacillus licheniformis xcex1-amylase is that it is a metalloprotein, requiring calcium ion for maximum stability of the enzymatically active configuration. Calcium binding increases resistance to denaturation at extremes of temperature and pH, but the ion is not involved in the starch hydrolysis reaction. Calcium salts are normally added to starch slurry for enzymatic liquification at, for example, 80 mg calcium ion/kg enzyme. Addition of calcium ion reduces enzyme cost but increases product refining cost. Therefore, a more stable enzyme would reduce the amount of calcium ion that must be added for enzyme stabilization and would thereby reduce costs of its removal later. Further, a more stable enzyme would reduce the required amount of the enzyme itself, under current reaction conditions; or, alternatively, higher temperatures could be used to shorten reaction times.
A principal reason that the starch industry has difficulties in achieving 96% DX is that a major by-product in the current starch-to-glucose processes is maltulose, a disaccharide consisting of an xcex1-1,4-glucosidic linkage between glucose and fructose (4-alpha-glucopyranosyl-D-fructose). Maltulose is formed by a nonenzymatic reaction during starch hydrolysis and is resistant to glucoamylase hydrolysis; and therefore, its formation reduces final glucose yields.
A recent study shows (Shetty, J. K. and Allen, W. G., 1988, supra) that maltulose formation is eliminated when liquifications are carried out below pH 6.0. (e.g., in the range of pH 5.0 to 5.8). In the same study, the stabilities of the four commercially available xcex1-amylases at pH 5.8 and 95xc2x0 C., the current liquification temperature, were compared. The highest thermostability was exhibited by an xcex1-amylase called TT-II, from a particular strains of Bacillus licheniformis. Under process conditions except for the absence of substrate, with 10 mg/kg calcium ion, TT-II lost about 10% of its activity in 25 minutes; in the presence of substrate (35% starch and 25 mg/kg added calcium ion), this time was extended to slight over 3 hours. These results imply half-lives at 95xc2x0 C. of about 2 hours and about 16 hours at 95xc2x0 C. in the absence or presence of substrate, respectively, for TT-II, the best commercially available xcex1-amylase for starch liquification.
As noted above, the liquification step of starch hydrolysis reduces viscosity of the hydrolysate and produces a high yield of oligosaccharide dextrins and some maltose. In saccharification, these maltodextrins and maltose are then further hydrolyzed to the final product, xcex1-D-glucose, by some other enzyme activity which may be a glucoamylase from some microbial source.
Other polysaccharide degrading enzymes with broad substrate specificity are known, for example, a pullulanase, also known as a debranching enzyme, which attacks xcex1-1,6-glycosidic linkages and can completely hydrolyze various polysaccharides into maltotriose (the microbial polysaccharide pullulan, for example). In principle, such an enzyme could be used in combination with other amylolytic activities for development of new approaches to complete hydrolysis of starch, provided that a proper combination of thermostable activities could be obtained to produce at least 96% DX.
For example, a thermophilic bacterium known as Thermoanaerobacter finnii has been shown to produce thermostable amylase and pullulanase activities which degrade starch completely to maltose (Antranikian, G., 1989, App. Biochem. and Biotech., 20/21:267-279). These enzymes were produced extracellularly, in the culture medium, a critical advantage for economical production of enzymes on the scale needed for commercial starch hydrolysis. An additional activity needed to complete the conversion of maltose to xcex1-D-glucose, a maltase, was also produced by this organism but only intracellularly. Further, the thermal stability of these enzymes was typified by temperature optima in the range of only 70xc2x0 C. to 90xc2x0 C. at pH 5.5, thus affording only marginal performance at the customary temperatures for starch hydrolysis.
An extracellular maltase from the fungus Paecilomyces Varioti has been reported (O""Mahony, M. R., et al., 1987, Biotechnology Letters 9:317-322). It has a pH optimum between 3.5 and 4.0, a temperature optimum of 60xc2x0 C., and a half-life at 60xc2x0 C. of about 8 hours. Accordingly, this enzyme offers little operational advantage over the intracellular maltase of T. finnii described above (Antranikian, G., 1989, supra).
Another frequently reported enzyme activity with potential for starch hydrolysis is that called xcex1-glucosidase. xcex1-glucosidases from various microbial sources exhibit significant diversity in their glucoside substrate specificities. Additionally, these enzymes can be either intracellular, extracellular, or membrane-bound. Thus, classification of these enzymes and functional comparisons between the xcex1-glucosidases from different sources are both difficult problems. Recently, Kelly and Fogerty (1983, Process Biochm. 18:6-12) have proposed a reclassification of bacterial xcex1-glucosidase enzymes into those with highest specific activity toward maltose and those with highest activity toward aryl-D-glucopyranosides.
In principle, an xcex1-glucosidase could be used to help completely digest the products of other starch liquification and saccharification enzymes, to produce high yields of xcex1-D-glucose in novel commercial processes. For example, an xcex1-glucosidase with the proper substrate preference might be used to digest maltose produced by a pullulanase or other enzymes. However, as noted in a recent study (Giblin, M. et al., 1987, Can. J. Microbiol. 33:614-18) typical xcex1-glucosidases from microbial sources are only slightly resistant to heat. In that same report (Giblin, id.), a novel xcex1-glucosidase from the thermophilic bacterium Bacillus caldovelox was described. This had a temperature optimum of about 50xc2x0 C. to 60xc2x0 C. at pH 5.6 to 6.0 and a half-life of about one hour at 70xc2x0 C. This enzyme was claimed to be the most thermostable microbial xcex1-glucosidase reported at that time. On the other hand, this enzyme was produced only intracellularly. Further, it showed rather narrow substrate specificity, with its highest specific activity being toward aryl-D-glucopyranosides rather than maltose. Also, this enzyme was unable to attack isomaltose.
Accordingly, there is a continuing need for a variety of microbial starch degrading enzymes which are more thermostable than those presently available, and which can be produced extracellularly to facilitate isolation on the large scale needed for commercial conversion of starch to glucose.
Another process with considerable commercial potential for utilization of thermostable polysaccharide depolymerizing enzymes is the hydrolysis of cellulose, the fibrous matter of all plant tissues. Complete digestion of cellulose to simple sugars requires synergistic action of three types of enzymes: endo-1,4-xcex2-cellulases; exo-1,4-xcex2-glucosidases; and xcex2-D-glucosidases. The latter release D-glucose from soluble cellodextrins and other glycosides including D-cellobiose, a major product of initial cellulose depolymerization steps.
A recent report (Tucker, M. P., et al., 1989, Bio/Technology 7:817-820) on so-called xe2x80x9cultra-thermostablexe2x80x9d cellulases from Acidothermus cellulolyticus, a thermophilic bacterium from a hot spring in Yellowstone Park, described an extracellular cellulase enzyme complex that produces cellobiose as the final product. An intracellular xcex2-glucosidase from the same source was used for production of the ultimate product, -D-glucose. This latter enzyme dramatically lost activity after reaching 75xc2x0 C., with the result that practically no activity could be assayed at 90xc2x0 C. at pH 5.0. In contrast, the two distinct extracellular cellulase activities showed biphasic decay curves upon heating in the absence of substrate, with optima of 75xc2x0 C. and 83xc2x0 C. and half-lives of about 12 minutes and less than 3 minutes, respectively, at 90xc2x0 C. and pH 5.0. A comparative survey of the literature was used to suggest that these cellulases, in particular, possess the highest temperature tolerance reported to date for this class of enzymes. Evidently, the efficiency of commercial hydrolysis of cellulose, for a variety of purposes, is highly constrained by a lack of enzymes with greater thermostability.
Saccharification (i.e., polysaccharide depolymerizing) enzymes from hyperthermophilic bacteria do not appear to be known in the art. Accordingly, a major object of the present invention is to provide methods for producing polysaccharide saccharification enzyme preparations, obtainable from hyperthermophilic bacteria such as P. furiosus, produced either extracellularly or intracellularly.
It is also an object of the present invention to provide cell-free and purified polysaccharide depolymerizing enzymes with thermostable activities that are highly desirable for various industrial applications.
The present invention is based, at least in part, on the finding that hyperthermophilic bacteria, for example, Pyrococcus furiosus, can be continuously cultivated at temperatures approaching 100xc2x0 C. (e.g., 97-99xc2x0 C.), to provide highly desirable enzymes at a useful rate. Further, it is based on the recognition of the highly unusual, if not unique, metabolic demands placed upon any organism which inhabits an environment that is nearly bereft of other living things and, hence, has little organic matter from which to obtain complex nutrients.
Thus, the present inventors have considered the possibility that Pyrococcus furiosus and other organisms sharing similar environments may have acquired efficient capabilities to digest whatever complex substrates, such as polysaccharides, for instance, that may be available and are otherwise relatively stable in the ocean depths at high temperature. These ambient polysaccharides might be expected to include, for example, cellulose and starch from plant vegetation and, perhaps, chitin from crustaceans.
Accordingly, the present inventors have found that P. furiosus produces several starch hydrolyzing enzymes, both extracellularly and intracellularly, which retain their activity for several hours at or above 100xc2x0 C. These comprise an xcex1-glucosidase, which has been highly purified, and amylolytic enzymes including an amylase and a pullulanase. In addition, at least one thermostable enzyme activity from P. furiosus that is useful in the process of degradation of cellulose to D-glucose has also been found, namely a xcex2-glucosidase, which is also active up to at least 95xc2x0 C. Furthermore, addition of appropriate carbohydrate substrates to cultures of P. furiosus has been found to induce enhancement of both extracellular and intracellular production of saccharification enzymes, usually those activities involved in degradative metabolism of the particular added substrate. The present invention thus enables production of such extremely thermostable saccharification enzymes for industrial applications, either directly from hyperthermophilic bacteria such as Pyrococcus furiosus, or from other more easily cultivated microorganisms through the use of genetic engineering technology that is now well known in the art.
Thus, in one aspect the present invention relates to a cell-free enzyme preparation obtainable from a hyperthermophilic bacterium, for example, Pyrococcus furiosus, having activities of the following enzymes: an amylase, a pullulanase, an xcex1-glucosidase and a xcex2-glucosidase. Each of these activities is distinguishable by its substrate specificity and thermal stability. This preparation is obtainable from P. furiosus by a process comprising the steps of cultivating cells of the bacterium in nutrient medium, collecting the cells from the medium, disrupting the cells, and removing the resulting insoluble cell debris, which results in a cell-free extract that contains these enzymes.
Culture medium of Pyrococcus furiosus also contains a variety of saccharification enzyme activities, the levels of which vary according to the composition of the nutrient medium. Their presence in the medium evidently results from extracellular enzyme production. Accordingly, the enzyme preparation of the present invention is also obtainable from Pyrococcus furiosus by a process comprising the steps of cultivating cells of the bacterium in nutrient medium and removing the cells from the medium, which results in cell-free medium that contains these enzymes.
The products resulting from the action of the enzyme preparation of this invention on various saccharide, as determined by thin-layer chromatography, indicate that this preparation is able to produce monomeric and oligomeric saccharides from starch, pullulan and glycogen. The preparation is able to convert a substantial portion of the starch completely to monomer after 72 hours at 85xc2x0 C. Although some oligomeric intermediates remain under the limited range of test conditions that have been tried, the apparent lack of accumulation of any particular intermediate indicates that, under proper conditions, the enzymes in the preparation of this invention can completely digest starch to glucose (i.e., achieve at least 96% DX). The major product from digestion of pullulan is a trimer (maltotriose). Only a minor amount of monomeric product is visible in dextran samples exposed to this enzyme preparation
According to a principal embodiment of this aspect of the present invention, the strain of Pyrococcus furiosus used for the cell-free enzyme preparation is derived from the deposit DSM 3638 of the Deutsche Sammlung von Mikroorganismen, Federal Republic of Germany.
In the enzyme preparation according to this invention, the activities of the amylase, pullulanase, and xcex1-glucosidase upon assay at pH 5.6 increase with increasing temperature in the range of 50xc2x0 C. to at least about 108xc2x0 C. Due to technical limitations of currently available methods for enzyme assays at temperatures substantially above the boiling point of water, that actual temperature optima for these enzyme activities have not been determined.
For comparison, as indicated in the Background, it is believed that the most extreme thermostability previously reported for a pullulanase activity, as well as for a maltase activity, was typified by temperature optima in the range of only 70xc2x0 C. to 90xc2x0 C. at pH 5.5 (Antranikian, G., 1989, App. Biochem. and Biotech., 20/21:267-279; O""Mahony, M. R., et al., 1987, Biotechnology Letters 9:317-322). For an xcex1-glucosidase, the most thermostable enzyme reported had a temperature optimum of about 50xc2x0 C. to 60xc2x0 C. at pH 5.6 to 6.0 and a half-life of about one hour at 70xc2x0 C. (Giblin, M. et al., 1987, Can. J. Microbiol. 33:614-18).
Further, in this enzyme preparation of the present invention, the half-life of the activity of the amylase at pH 5.6 and 98xc2x0 C., in the absence of substrate, with no added stabilizing agent, is about 4 hours; the half-life of the activity of the pullulanase at pH 5.6 and 98xc2x0 C., in the absence of substrate, with no added stabilizing agent, is about 7 hours; and the half-life of the activity of the xcex1-glucosidase at pH 5.6 and 98xc2x0 C., in the absence of substrate, with no added stabilizing agent, is about 8 hours.
It is known that cell-free enzyme preparations of the present invention contain thermostable proteolytic enzyme activities which are highly active at 98xc2x0 C. (see U.S. patent application Ser. No. 07/406,327, filed Sep. 12, 1989). Therefore, these activities may substantially reduce the half-lives of the present enzyme activities compared to the stabilities of the same enzymes that would be observed in the absence of this protease. (See, for example, the approximately 5 fold increase in thermostability of the xcex1-glucosidase upon purification, Example 3, below.)
Another aspect of the present invention relates to a purified xcex1-glucosidase obtainable from Pyrococcus furiosus and having activities on the following substrates at 95xc2x0 C. and pH 5.6, in descending order, as follows: isomaltose, maltose, methyl-xcex1-D-glucopyranoside, and p-nitrophenol-xcex1-D-glucopyranoside. This purified xcex1-glucosidase has a pH optimum between about pH 5.2 and pH 5.8 at 98xc2x0 C., and shows no significant loss of activity during assay at pH 5.6 over the temperature range of 105xc2x0 C. to 115xc2x0 C., the highest temperature that has been tested so far due to technical limitations of enzyme assays at elevated temperatures. The half-life of the activity of the purified xcex1-glucosidase at pH 5.6 and 98xc2x0 C., in the absence of substrate, with no added stabilizing agent, is about 40 hours. The xcex1-glucosidase of this invention has been purified at least 310-fold and comprises a single polypeptide having an approximate molecular mass value of about 125 kilodaltons (kDa) as determined by electrophoresis in polyacrylamide gels in the presence of SDS. Purification and characterization of this xcex1-glucosidase is described in detail in Example 3, below.
In yet another aspect, the present invention also relates to a cell-free preparation of xcex2-glucosidase from Pyrococcus furiosus having activity at 95xc2x0 C. and pH 5.6 that is greater on p-nitrophenol-xcex2-D-glucopyranoside than its activity on p-nitrophenol-xcex1-D-glucopyranoside. The majority of this xcex2-glucosidase is not bound to an anion exchange resin in the absence of salt under slightly basic pH conditions (e.g., at pH 8.5), as described in Example 3, below, indicating that at least one major component of this activity is vested in an acidic protein. Further, the xcex2-glucosidase activity resides in at least two molecularly distinct species, one of which has a relative mobility about 2.4 times higher than that of the xcex1-glucosidase of the present invention, upon electrophoresis under nondenaturing conditions, and the other of which has a relative mobility about 7.0 times lower than that of this same xcex1-glucosidase under the same conditions.
This invention also relates to a process for producing a cell-free enzyme preparation from Pyrococcus furiosus having activities of the following enzymes: an amylase, a pullulanase, an xcex1-glucosidase and a xcex2-glucosidase, comprising the steps of: cultivating cells of the bacterium in nutrient medium at a temperature of about 98xc2x0 C. under an anaerobic atmosphere at ambient pressure, under conditions of continuous steady-state culture, and where the nutrient medium comprises sea water containing complex carbon sources, and optionally, elemental sulfur; and separating said cells from said medium, which results in cell-free medium that contains the enzymes. Further, optionally, the enzymes inside the cells are prepared by the additional steps of disrupting the cells and removing the resulting insoluble cell debris, which results in a cell-free extract that contains said enzymes.
This process of for obtaining cell-free preparations of saccharification enzymes from a hyperthermophilic bacterium, such as Pyrococcus furiosus, for instance, is advantageously coupled with a method for continuous steady-state culture of such organisms which has been developed by the present inventors (Example 1). The yields per unit of culture medium and the reductions in labor and downtime for equipment cleaning of this approach compare quite favorably to the more usual batch approaches for cultivation of bacteria which grow only at lower temperatures.
An embodiment of this overall enzyme production method which incorporates this simple continuous culture method is described herein for producing saccharification enzymes, in which advantageously the cultivating of the Pyrococcus furiosus is at a temperature of about 98xc2x0 C. under an anaerobic atmosphere at ambient pressure, under conditions of continuous steady-state culture, and where the nutrient medium comprises sea water containing complex carbon sources, and optionally, for better growth, elemental sulfur.
The present inventors have also discovered that addition of various oligosaccharides or polysaccharides to a culture of P. furiosus enhances production of saccharification enzymes. Therefore, the present invention also relates to the process for producing a cell-free enzyme preparation described above where the nutrient medium further comprises an amount of carbohydrate substrate effective for enhancing production of any of the enzyme activities described above. Evidently, substrates with (1,4) glucose linkages enhance production of all the saccharification enzymes in the preparation of this invention, including the xcex2-glucosidase, although different substrates have differential effects on the different enzymes and their relative distributions inside the cells or in the medium. Advantageously for enhancement of total enzyme production, this substrate is maltose, pullulan, starch or glycogen.
In a most preferred embodiment of this process, the nutrient medium further comprises an amount of polysaccharide substrate effective for enhancing production of any of the enzymes in the culture medium. Most advantageously, this substrate is starch or glycogen.
Thus, compared to using complex carbon sources (yeast extract and tryptone) with elemental sulfur, addition of maltose, starch or glycogen enhances production of the total level of each of the three saccharification enzyme activities, amylase, pullulanase, and xcex1-glucosidase, in either the cells or the culture medium or both, by factors ranging from about 3 to 50 fold. The best yields per liter of culture of each enzyme obtained so far, with substrate added to the culture as indicated, are as follows: amylase (glycogen, enzyme in medium) 60,100 units; pullulanase (maltose, intracellular) 8020 units; and xcex1-glucosidase (glycogen, intracellular) 9584; xcex2-glucosidase (starch, intracellular) 220 units.
The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Figures and Examples included therein.